An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells

Embryonic stem cells (ESCs), with their dual capacity to self-renew and differentiate, are commonly used to study differentiation, epigenetic regulation, lineage choices, and more. Using non-directed retroviral integration of a YFP/Cherry exon into mouse ESCs, we generated a library of over 200 endogenously tagged fluorescent fusion proteins and present several proof-of-concept applications of this library. We show the utility of this library to track proteins in living cells; screen for pluripotency-related factors; identify heterogeneously expressing proteins; measure the dynamics of endogenously labeled proteins; track proteins recruited to sites of DNA damage; pull down tagged fluorescent fusion proteins using anti-Cherry antibodies; and test for interaction partners. Thus, this library can be used in a variety of different directions, either exploiting the fluorescent tag for imaging-based techniques or utilizing the fluorescent fusion protein for biochemical pull-down assays, including immunoprecipitation, co-immunoprecipitation, chromatin immunoprecipitation, and more. Keywords: embryonic stem cells; imaging; live imaging; fluorescence; differentiation; pluripotency; GFP; microscopy; DNA damage; protein dynamicsNational Institutes of Health (U.S.) (Grant HD045022)National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant R01NS088538-01

Heterochromatin Protein 1β (HP1β) has distinct functions and distinct nuclear distribution in pluripotent versus differentiated cells

Background: Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into every cell type and to self-renew. These characteristics correlate with a distinct nuclear architecture, epigenetic signatures enriched for active chromatin marks and hyperdynamic binding of structural chromatin proteins. Recently, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, yet the role of the major heterochromatin proteins in pluripotency is unknown. Results: Here we identify Heterochromatin Protein 1β (HP1β) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1β is differentially localized and differentially associated with chromatin. Deletion of HP1β, but not HP1aα, in ESCs provokes a loss of the morphological and proliferative characteristics of embryonic pluripotent cells, reduces expression of pluripotency factors and causes aberrant differentiation. However, in differentiated cells, loss of HP1β has the opposite effect, perturbing maintenance of the differentiation state and facilitating reprogramming to an induced pluripotent state. Microscopy, biochemical fractionation and chromatin immunoprecipitation reveal a diffuse nucleoplasmic distribution, weak association with chromatin and high expression levels for HP1β in ESCs. The minor fraction of HP1β that is chromatin-bound in ESCs is enriched within exons, unlike the situation in differentiated cells, where it binds heterochromatic satellite repeats and chromocenters. Conclusions: We demonstrate an unexpected duality in the role of HP1β: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1β function both depends on, and regulates, the pluripotent state

[Retracted] Peroxisome proliferator-activated receptor β/δ induces myogenesis by modulating myostatin activity

Classically, peroxisome proliferator-activated receptor β/δ (PPARβ/δ) function was thought to be restricted to enhancing adipocyte differentiation and development of adipose-like cells from other lineages. However, recent studies have revealed a critical role for PPARβ/δ during skeletal muscle growth and regeneration. Although PPARβ/δ has been implicated in regulating myogenesis, little is presently known about the role and, for that matter, the mechanism(s) of action of PPARβ/δ in regulating postnatal myogenesis. Here we report for the first time, using a PPARβ/δ-specific ligand (L165041) and the PPARβ/δ-null mouse model, that PPARβ/δ enhances postnatal myogenesis through increasing both myoblast proliferation and differentiation. In addition, we have identified Gasp-1 (growth and differentiation factor-associated serum protein-1) as a novel downstream target of PPARβ/δ in skeletal muscle. In agreement, reduced Gasp-1 expression was detected in PPARβ/δ-null mice muscle tissue. We further report that a functional PPAR-responsive element within the 1.5-kb proximal Gasp-1 promoter region is critical for PPARβ/δ regulation of Gasp-1. Gasp-1 has been reported to bind to and inhibit the activity of myostatin; consistent with this, we found that enhanced secretion of Gasp-1, increased Gasp-1 myostatin interaction and significantly reduced myostatin activity upon L165041-mediated activation of PPARβ/δ. Moreover, we analyzed the ability of hGASP-1 to regulate myogenesis independently of PPARβ/δ activation. The results revealed that hGASP-1 protein treatment enhances myoblast proliferation and differentiation, whereas silencing of hGASP-1 results in defective myogenesis. Taken together these data revealed that PPARβ/δ is a positive regulator of skeletal muscle myogenesis, which functions through negatively modulating myostatin activity via a mechanism involving Gasp-1

Embryonic stem cell differentiation is regulated by SET through interactions with p53 and β-Catenin

The multifunctional histone chaperone, SET, is essential for embryonic development in the mouse. Previously, we identified SET as a factor that is rapidly downregulated during embryonic stem cell (ESC) differentiation, suggesting a possible role in the maintenance of pluripotency. Here, we explore SET's function in early differentiation. Using immunoprecipitation coupled with protein quantitation by LC-MS/MS, we uncover factors and complexes, including P53 and β-catenin, by which SET regulates lineage specification. Knockdown for P53 in SET-knockout (KO) ESCs partially rescues lineage marker misregulation during differentiation. Paradoxically, SET-KO ESCs show increased expression of several Wnt target genes despite reduced levels of active β-catenin. Further analysis of RNA sequencing datasets hints at a co-regulatory relationship between SET and TCF proteins, terminal effectors of Wnt signaling. Overall, we discover a role for both P53 and β-catenin in SET-regulated early differentiation and raise a hypothesis for SET function at the β-catenin-TCF regulatory axis.Published versio

Negative Auto-Regulation of Myostatin Expression is Mediated by Smad3 and MicroRNA-27

<div><p>Growth factors, such as myostatin (Mstn), play an important role in regulating post-natal myogenesis. In fact, loss of Mstn has been shown to result in increased post-natal muscle growth through enhanced satellite cell functionality; while elevated levels of Mstn result in dramatic skeletal muscle wasting through a mechanism involving reduced protein synthesis and increased ubiquitin-mediated protein degradation. Here we show that miR-27a/b plays an important role in feed back auto-regulation of Mstn and thus regulation of post-natal myogenesis. Sequence analysis of <i>Mstn</i> 3′ UTR showed a single highly conserved miR-27a/b binding site and increased expression of miR-27a/b was correlated with decreased expression of <i>Mstn</i> and vice versa both <i>in vitro</i> and in mice <i>in vivo</i>. Moreover, we also show that <i>Mstn</i> gene expression was regulated by miR-27a/b. Treatment with miR-27a/b-specific AntagomiRs resulted in increased <i>Mstn</i> expression, reduced myoblast proliferation, impaired satellite cell activation and induction of skeletal muscle atrophy that was rescued upon either blockade of, or complete absence of, Mstn. Consistent with this, miR-27a over expression resulted in reduced <i>Mstn</i> expression, skeletal muscle hypertrophy and an increase in the number of activated satellite cells, all features consistent with impaired Mstn function. Loss of <i>Smad3</i> was associated with increased levels of Mstn, concomitant with decreased miR-27a/b expression, which is consistent with impaired satellite cell function and muscular atrophy previously reported in <i>Smad3</i>-null mice. Interestingly, treatment with Mstn resulted in increased miR-27a/b expression, which was shown to be dependent on the activity of Smad3. These data highlight a novel auto-regulatory mechanism in which Mstn, via Smad3 signaling, regulates miR-27a/b and in turn its own expression. In support, Mstn-mediated inhibition of <i>Mstn</i> 3′ UTR reporter activity was reversed upon miR-27a/b-specific AntagomiR transfection. Therefore, miR-27a/b, through negatively regulating <i>Mstn</i>, plays a role in promoting satellite cell activation, myoblast proliferation and preventing muscle wasting.</p></div

AntagomiR-mediated inhibition of miR-27a enhances endogenous Mstn expression and function <i>in vivo</i>.

<p>(A) qPCR analysis of <i>Mstn</i> mRNA expression in TA muscle isolated from WT mice (n = 3) 8 days post intramuscular injection of either AntagomiR Neg or AntagomiR-27a. <i>p</i><0.05 (*). (B) Representative images of H&E stained AntagomiR Neg and AntagomiR-27a injected TA muscle from WT mice. Scale bars = 100 µm. (C) Graph showing average myofiber CSA (µm²) in AntagomiR Neg and AntagomiR-27a injected TA muscle from WT mice. Average myofiber area was calculated from 10 random images per coverslip (n = 3). (D) Frequency distribution of myofiber area (µm²) in AntagomiR Neg and AntagomiR-27a injected TA muscle from WT mice as calculated from 10 random images per coverslip (n = 3). (E) <i>Left</i>: Representative merged immunofluorescence image showing a Pax7⁺ cell (Green; white arrowhead) in an AntagomiR Neg injected TA muscle cross section from WT mice. Sections were also stained for Laminin (Red) and nuclei were counterstained with DAPI (Blue). Scale bar = 10 µm. <i>Right</i>: Graph showing the number of Pax7⁺ cells in AntagomiR Neg and AntagomiR-27a injected TA muscle from WT mice. Bars represent mean number ± S.E.M of Pax7⁺ cells, per 100 myofibers, from 3 sections each collected from AntagomiR Neg and AntagomiR-27a injected WT mice (n = 3). <i>p</i><0.01 (**). (F) Graph showing the number of MyoD⁺ cells in AntagomiR Neg and AntagomiR-27a injected TA muscle from WT mice. Bars represent mean number ± S.E.M of MyoD⁺ cells, per 100 myofibers, from 3 sections each collected from AntagomiR Neg and AntagomiR-27a injected WT mice (n = 3). <i>p</i><0.001 (***).</p

miR-27a/b targets and represses <i>Mstn</i> expression.

<p>(A) <i>In silico</i> analysis, using TargetScan algorithms, showing a 8 mer seed match (grey box) between murine miR-27a (mmu-miR-27a) and miR-27b (mmu-miR-27b) and the miR-27a/b binding site located within the <i>Mstn</i> 3′ UTR sequence (mmu-<i>Mstn</i> 3′UTR). (B) Assessment of pMIR-REPORT™ luciferase activity in C2C12 myoblasts co-transfected with the <i>Mstn</i> 3′UTR reporter construct (<i>Mstn</i> 3′UTR) and either control (pcDNA-miR-neg), miR-27a over expression construct (pcDNA-miR-27a), negative control AntagomiR (AntagomiR Neg) or a miR-27a-specific AntagomiR (AntagomiR-27a) for 48h. (C) Assessment of pMIR-REPORT™ luciferase activity in C2C12 myoblasts co-transfected with the mutant <i>Mstn</i> 3′UTR reporter construct (<i>Mstn</i> 3′UTR-mut), where the miR-27a/b binding site has been mutated, and either control (pcDNA-miR-neg), miR-27a over expression construct (pcDNA-miR-27a), negative control AntagomiR (AntagomiR Neg) or a miR-27a-specific AntagomiR (AntagomiR-27a) for 48h. For all pMIR-REPORT™ transfections, luciferase activity was normalized to Renilla luciferase and expressed as fold change relative to control (pcDNA-miR-neg). Bars represent mean values ± S.E.M (n = 3). <i>p</i><0.05 (*) and <i>p</i><0.001(***). qPCR analysis of <i>Mstn</i> mRNA expression (D) and precursor-miR-27a/b (pre-miR-27a/b) expression (E) in Heart, Liver, <i>M. Biceps femoris</i> muscle (BF) and <i>M. Soleus muscle</i> (Sol.) collected from 4-week-old wild type (WT) mice. Bars represent fold change (relative to Heart) ± S.E.M (n = 3) normalized to <i>GAPDH</i> (D) or U6 (E) expression. <i>p</i><0.05 (*) and <i>p</i><0.001(***). qPCR analysis of (F) <i>Mstn</i> and (G) miR-27b expression in C2C12 myoblast cultures differentiated across a time course (24 h, 48 h, 72 h and 96 h differentiation). Bars represent fold change (relative to 24 h control) ± S.E.M (n = 3) normalized to either <i>GAPDH</i> (F) or U6 (G) expression. <i>p</i><0.001(***).</p

Increased <i>Mstn</i> expression in <i>Smad3</i>-null mice is due to reduced miR-27a/b expression.

<p>qPCR analysis of (A) <i>Mstn</i>, (B) miR-27a and (C) miR-27b expression in <i>M. Tibialis anterior</i> muscle (TA), <i>M. Gastrocnemius</i> muscle (GAS) and <i>M. Quadriceps muscle</i> (QUAD) isolated from WT and <i>Smad3</i>-null mice. Bars represent fold change (relative to respective WT control) ± S.E.M (n = 3) normalized to either <i>GAPDH</i> (A) or U6 (B & C) expression. <i>p</i><0.001 (***). (D) qPCR analysis of <i>Mstn</i> expression in 48 h differentiated C2C12 myotubes treated without (0.05% DMSO) or with SIS3 (10 µM) for 24 h. <i>p</i><0.001 (***). (E) qPCR analysis of pre-miR-27a/b expression in 48 h differentiated C2C12 myotubes treated without (0.05% DMSO) or with SIS3 (10 µM) for 24 h. <i>p</i><0.001 (***). (F) qPCR analysis of <i>Mstn</i> in 72 h differentiated primary myoblast cultures isolated from WT and <i>Smad3</i>-null mice that were transfected with either non targeting miRNA negative control (miRNA neg control) or miR-27b-specific mimic (miR-27b mimic). Bars represent fold change (relative to WT miRNA Neg control transfected myoblasts) ± S.E.M (n = 3) normalized to <i>GAPDH</i> expression. <i>p</i><0.01 (**) and <i>p</i><0.001 (***).</p

Mstn treatment up regulates miR-27a/b expression via Smad3 to negatively auto-regulate it's own expression.

<p>qPCR analysis of pre-miR-27a/b expression in C2C12 myoblasts (A) and 48 h differentiated C2C12 myotubes (B) following 12 h treatment with conditioned medium from either control CHO cells (CCM) or from CHO-cells designed to produce and secrete Mstn protein (CMM). Bars represent fold change (relative to respective CCM control) ± S.E.M (n = 3) normalized to U6 expression. <i>p</i><0.05 (*) and <i>p</i><0.01 (**). (C) Assessment of miR-27a and miR-27b promoter-reporter luciferase activity in C2C12 myoblasts transfected with the miR-27a promoter (miR-27a pro), miR-27b promoter (miR-27b pro) or a mutant miR-27b promoter reporter construct, where the smad binding site has been mutated (miR-27b pro-mut). Transfected C2C12 myoblasts were treated without (CCM) or with CMM in the absence (0.05% DMSO) or presence of SIS3 (10 µM) for 24 h prior to assessment of luciferase activity. All luciferase activity was normalized to Renilla luciferase and expressed as fold change relative to respective controls (CCM+DMSO). Bars represent mean values ± S.E.M (n = 3). <i>p</i><0.05 (*), <i>p</i><0.01 (**) and <i>p</i><0.001 (***). (D) Assessment of pMIR-REPORT™ luciferase activity in C2C12 myoblasts co-transfected with <i>Mstn</i> 3′UTR and either AntagomiR Neg, AntagomiR-27a or AntagomiR-27b in the absence (−) or presence (+) of CMM. Bars represent mean values ± S.E.M (n = 3). <i>p</i><0.001 (***). (E) Assessment of pMIR-REPORT™ luciferase activity in C2C12 myoblasts co-transfected with <i>Mstn</i> 3′UTR-mut and either AntagomiR Neg, AntagomiR-27a or AntagomiR-27b in the absence (−) or presence (+) of Mstn protein (CMM). Bars represent mean values ± S.E.M (n = 3). All luciferase activity was normalized to Renilla luciferase and expressed as fold change relative to control (CMM - and AntagomiR Neg +). (F) Based on the data presented in this current manuscript we propose that upon Mstn-mediated receptor activation Smad3 up-regulates the expression of miR-27a/b, which in turn leads to reduced <i>Mstn</i> expression and impaired Mstn function, thus forming the basis of a novel negative Mstn auto-regulatory loop in muscle.</p

Muscle-specific microRNA1 (miR1) targets heat shock protein 70 (HSP70) during dexamethasone-mediated atrophy

High doses of dexamethasone (Dex) or myostatin (Mstn) induce severe atrophy of skeletal muscle. Here we show a novel microRNA1 (miR1)-mediated mechanism through which Dex promotes skeletal muscle atrophy. Using both C2C12 myotubes and mouse models of Dex-induced atrophy we show that Dex induces miR1 expression through glucocorticoid receptor (GR). We further show that Mstn treatment facilitates GR nuclear translocation and thereby induces miR1 expression. Inhibition of miR1 in C2C12 myotubes attenuated the Dex-induced increase in atrophy-related proteins confirming a role for miR1 in atrophy. Analysis of miR1 targets revealed that HSP70 is regulated by miR1 during atrophy. Our results demonstrate that increased miR1 during atrophy reduced HSP70 levels, which resulted in decreased phosphorylation of AKT, as HSP70 binds to and protects phosphorylation of AKT. We further show that loss of pAKT leads to decreased phosphorylation, and thus, enhanced activation of FOXO3, up-regulation of MuRF1 and Atrogin-1, and progression of skeletal muscle atrophy. Based on these results, we propose a model whereby Dex- and Mstn-mediated atrophic signals are integrated through miR1, which then either directly or indirectly, inhibits the proteins involved in providing protection against atrophy